The invention pertains to human N-type calcium channel xcex11B subunit isoforms.
Voltage gated calcium channels, also known as voltage dependent calcium channels (VDCCs) are multisubunit membrane spanning proteins which permit controlled calcium influx from an extracellular environment into the interior of a cell. Several types of voltage gated calcium channel have been described in different tissues, including N-type, P/Q-type, L-type and T-type channels. A voltage gated calcium channel permits entry into the cell of calcium upon depolarization of the membrane of the cell, which is a lessening of the difference in electrical potential between the outside and the inside of the cell.
A voltage gated calcium channel contains several proteins, including xcex11, xcex12, xcex2, and xcex3 subunits. Subtypes of the calcium channel subunits also are known. For instance, xcex11 subtypes include xcex11A, xcex11B, xcex11C, xcex11D, xcex11E and xcex11S. Each subunit may have one or more isoforms which result from alternative splicing of RNA in the formation of a completed messenger RNA which encodes the subunit. For example, at least four isoforms of the rat N-type xcex11B subunit are known (see, e.g., Lin et al., Neuron 18:153-166, 1997).
Isoforms of calcium channel xcex11 subunits may be expressed differently in different tissues (see, e.g., Lin et al., 1997). Differential expression of subunits isoforms raises the possibility of developing therapeutics which are specific for distinct isoforms of the xcex11 subunits, thereby lessening side effects resulting from the use of therapeutics which are effective for more than one calcium channel isoform. Two isoforms of the human N-type calcium channel xcex11B subunit were published by Williams et al in 1992 (Science 257:389-395). Given the existence of several additional rat isoforms in a highly conserved gene family, it is surprising that additional human isoforms of the N-type calcium channel xcex11B subunit have not been discovered. Such isoforms would be useful for developing isoform-specific therapeutics.
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and inhibitors of the foregoing nucleic acids and polypeptides which reduce voltage-gated calcium influx. The foregoing can be used in the diagnosis or treatment of conditions characterized by increased or decreased human N-type calcium channel hxcex11B+SFVG subunit activity and can be used in methods in which it is therapeutically useful to increase or decrease human N-type calcium channel hxcex11B+SFVG subunit activity such as treatments for stroke, pain (e.g., neuropathic pain), traumatic brain injury and conditions characterized by increased or decreased voltage regulated calcium influx. Here, we present the identification of a novel human N-type calcium channel xcex11B subunit, hxcex11B+SFVG, which plays a role in voltage-gated calcium influx.
It was discovered that a brain xcex11B calcium channel subunit isoform (splice variant) contains a four amino acid insert relative to published human xcex11B calcium channel isoforms (SEQ ID NO:5 [GenBank accession number M94172], SEQ ID NO:7 [GenBank accession number M94173]). Surprisingly, this insert, SFVG (SEQ ID NO:2, encoded by SEQ ID NO:1), is similar but not identical to an insert found in a rat xcex11B channel (GenBank accession number M92905). A significant proportion of the human N-type calcium channel xcex11B subunit mRNA in brain was found to be the hxcex11B+SFVG sub-type; given the abundance of its expression the isolation of this sub-type so long after the identification of other xcex11B isoforms is unexpected. The SFVG-containing human N-type calcium channel hxcex11B+SFVG subunit also lacks an amino acid sequence, ET, which is present in published human N-type calcium channel hxcex11B+SFVG subunit isoforms (amino acids 1557-1558 of SEQ ID NOs:5 and 7).
The invention involves in one aspect an isolated human N-type calcium channel xcex11B subunit polypeptide which includes the amino acid sequence of SEQ ID NO:2 (an hxcex11B+SFVG polypeptide). In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:4, and preferably consists of the amino acid sequence of SEQ ID NO:4. In another embodiment the hxcex11B+SFVG calcium channel polypeptide is a fragment or variant of the foregoing polypeptides, wherein the fragment or variant includes the amino acid sequence of SEQ ID NO:2 or additions, deletions or substitutions thereof which confer the same function as SEQ ID NO: 2. Preferred variants include those having additions, substitutions or deletions relative to the human N-type calcium channel hxcex11B+SFVG subunit polypeptide sequence disclosed herein, particularly those variants which retain one or more of the activities of the human N-type calcium channel hxcex11B+SFVG subunit, including subunits with or without the ET exon sequence.
According to another aspect of the invention, an isolated nucleic acid molecule which encodes any of the foregoing human N-type calcium channel hxcex11B+SFVG subunit polypeptide is provided. In certain embodiments, the nucleic acid molecule includes SEQ ID NO:1. In one preferred embodiment, the human N-type calcium channel hxcex11B+SFVG subunit polypeptides is encoded by a nucleic acid molecule which comprises the nucleotide sequence of SEQ ID NO:3 (Williams et al. sequence +SFVG, xe2x88x92ET), and which preferably consists of the nucleotide sequence of SEQ ID NO:3. In another embodiment the nucleic acid is an allele of the nucleic acid sequence of SEQ ID NO:3.
In another aspect the invention is an expression vector comprising the human N-type calcium channel hxcex11B+SFVG subunit nucleic acid molecule operably linked to a promoter. Also included within the invention is a host cell transformed or transfected with the expression vector.
According to another aspect of the invention, an agent which selectively binds the human N-type calcium channel hxcex11B+SFVG subunit polypeptide or a nucleic acid that encodes the human N-type calcium channel hxcex11B+SFVG subunit polypeptide is provided. By xe2x80x9cselectively bindsxe2x80x9d it is meant that the agent binds the human N-type calcium channel hxcex11B+SFVG subunit polypeptide or nucleic acid, or any fragment thereof which retains the amino acids of SEQ ID NO:2 or the nucleotides of SEQ ID NO:1, to a greater extent than the agent binds other human N-type calcium channel xcex11B subunit isoforms, and preferably does not bind other human N-type calcium channel xcex11B subunit isoforms. In one embodiment, the agent is a polypeptide which binds selectively to the human N-type calcium channel hxcex11B+SFVG subunit polypeptide. The polypeptide can be a monoclonal antibody, a polyclonal antibody, or an antibody fragment selected from the group consisting of a Fab fragment, a F(ab)2 fragment and a fragment including a CDR3 region. In another embodiment, the agent is an antisense nucleic acid which selectively binds to a nucleic acid encoding the human N-type calcium channel hxcex11B+SFVG subunit polypeptide. Preferably the foregoing agents are inhibitors (antagonists) or agonists of the calcium channel activity of the human N-type calcium channel hxcex11B+SFVG subunit polypeptide.
According to another aspect of the inventions, a dominant negative human N-type calcium channel hxcex11B+SFVG subunit polypeptide is provided. The dominant negative polypeptide is an inhibitor of the function of the calcium channel.
The invention also provides compositions including any of the foregoing polypeptides, nucleic acids or agents in combination with a pharmaceutically acceptable carrier.
In another aspect of the invention a method for inhibiting human N-type calcium channel hxcex11B+SFVG subunit activity in a mammalian cell is provided. The method involves the step of contacting the mammalian cell with an amount of a human N-type calcium channel hxcex11B+SFVG subunit inhibitor effective to inhibit calcium influx in the mammalian cell. Preferably the inhibitor is selected from the group consisting of a peptide or an antibody which selectively binds the human N-type calcium channel hxcex11B+SFVG subunit polypeptide, an antisense nucleic acid which binds a nucleic acid encoding human N-type calcium channel hxcex11B+SFVG subunit polypeptide and a dominant negative human N-type calcium channel hxcex11B+SFVG subunit polypeptide.
According to still another aspect the invention, a method for treating a subject having a stroke, pain (e.g., neuropathic pain), or traumatic brain injury is provided. The method involves the step of administering to a subject in need of such treatment an inhibitor of the human N-type calcium channel hxcex11B+SFVG subunit polypeptide in an amount effective to inhibit voltage regulated calcium influx. In another embodiment of the foregoing methods, the inhibitor is administered prophylactically to a subject at risk of having a stroke.
The human N-type calcium channel hxcex11B+SFVG subunit polypeptides and nucleic acids which encode such polypeptides are useful for increasing the amount of human N-type calcium channel hxcex11B+SFVG subunit polypeptides in a cell. Increasing the amount of human N-type calcium channel hxcex11B+SFVG subunit polypeptides in a cell results in increased voltage regulated calcium influx. This is useful where it is desired to increase the amount of voltage regulated calcium influx which is mediated by a human N-type calcium channel.
Thus according to another aspect of the invention, a method for increasing human N-type calcium channel hxcex11B+SFVG subunit expression in a cell is provided. The method involves the step of contacting the cell with a molecule selected from the group consisting of a human N-type calcium channel hxcex11B+SFVG subunit nucleic acid and a human N-type calcium channel hxcex11B+SFVG subunit polypeptide in an amount effective to increase voltage regulated calcium influx in the cell. In certain embodiments, the cell is contacted with one or more human N-type calcium channel non-hxcex11B+SFVG subunits, such as a xcex2 subunit, or nucleic acids encoding such non-hxcex11B+SFVG subunits.
According to another aspect of the invention, a method for increasing calcium channel voltage regulated calcium influx in a subject is provided. The method involves the step of administering to a subject in need of such treatment a molecule selected from the group consisting of a human N-type calcium channel hxcex11B+SFVG subunit nucleic acid and a human N-type calcium channel hxcex11B+SFVG subunit polypeptide in an amount effective to increase voltage regulated calcium influx in the subject.
According to a further aspect of the invention, a method for identifying lead compounds for a pharmacological agent useful in the treatment of disease associated with increased or decreased voltage regulated calcium influx mediated by a human N-type calcium channel is provided. A cell or other membrane-encapsulated space comprising a human N-type calcium channel hxcex11B+SFVG subunit polypeptide is provided. The cell or other membrane-encapsulated space preferably is loaded with a calcium-sensitive compound which is detectable in the presence of calcium. The cell or other membrane-encapsulated space is contacted with a candidate pharmacological agent under conditions which, in the absence of the candidate pharmacological agent, cause a first amount of voltage regulated calcium influx into the cell or other membrane-encapsulated space. A test amount of voltage regulated calcium influx then is determined. For example, in a preferred embodiment, fluorescence of a calcium-sensitive compound then is detected as a measure of the voltage regulated calcium influx. If the test amount of voltage regulated calcium influx is less than the first amount, then the candidate pharmacological agent is a lead compound for a pharmacological agent which reduces voltage regulated calcium influx. If the test amount of voltage regulated calcium influx is greater than the first amount, then the candidate pharmacological agent is a lead compound for a pharmacological agent which increases voltage regulated calcium influx.
In another aspect of the invention, methods for identifying compounds which selectively or preferentially bind a human N-type calcium channel hxcex11B+SFVG subunit isoform are provided. In one embodiment, the method includes providing a first cell or membrane encapsulated space which expresses a human N-type calcium channel hxcex11B+SFVG subunit isoform, and providing a second cell or membrane encapsulated space which expresses a human N-type calcium channel non-hxcex11B+SFVG subunit isoform, wherein the second cell or membrane encapsulated space is identical to the first cell except for the xcex11B isoform expressed. The first cell or membrane encapsulated space and the second cell or membrane encapsulated space are contacted with a compound, and the binding of the compound to the first cell or membrane encapsulated space and the second cell or membrane encapsulated space is determined. A compound which binds the first cell or membrane encapsulated space but does not bind the second cell or membrane encapsulated space is a compound which selectively binds the human N-type calcium channel hxcex11B+SFVG subunit isoform. A compound which binds the first cell or membrane encapsulated space in an amount greater than the compound binds the second cell or membrane encapsulated space is a compound which preferentially binds the human N-type calcium channel hxcex11B+SFVG subunit isoform. In another embodiment of the method, a human N-type calcium channel hxcex11B+SFVG subunit isoform polypeptide or nucleic acid and a human N-type calcium channel non-hxcex11B+SFVG subunit isoform polypeptide or nucleic acid are provided and contacted with a compound. The binding of the compound to the human N-type calcium channel hxcex11B+SFVG subunit isoform polypeptide or nucleic acid and the human N-type calcium channel non-hxcex11B+SFVG subunit isoform polypeptide or nucleic acid then is determined. A compound which binds the human N-type calcium channel hxcex11B+SFVG subunit isoform polypeptide or nucleic acid but does not bind the human N-type calcium channel non-hxcex11B+SFVG subunit isoform polypeptide or nucleic acid is a compound which selectively binds the human N-type calcium channel hxcex11B+SFVG subunit isoform polypeptide or nucleic acid. A compound which binds the human N-type calcium channel hxcex11B+SFVG subunit isoform polypeptide or nucleic acid in an amount greater than the human N-type calcium channel non-hxcex11B+SFVG subunit isoform polypeptide or nucleic acid is a compound which preferentially binds the human N-type calcium channel hxcex11B+SFVG subunit isoform polypeptide or nucleic acid. Also included in the invention are compounds identified using the foregoing methods.
According to another aspect of the invention, a method for selectively treating a subject having a condition characterized by aberrant brain neuronal calcium current is provided. The method includes the step of administering to a subject in need of such treatment a pharmacological agent which is selective for a human N-type calcium channel hxcex11B+SFVG subunit, in an amount effective to normalize the aberrant neuronal calcium current. Aberrant means a level of calcium current (calcium influx) which is outside of a normal range as understood in the medical arts. Normalize means that the calcium current is brought within the normal range.
Also presented herein is an identification of characteristics of certain calcium channel subunit isoforms with respect to voltage-dependent activation. It has been discovered, surprisingly, that the presence or absence of an exon comprising the amino acids ET is important for the kinetics of channel activation. Thus, in still other aspects of the invention, a variety of novel assays, screens, recombinant products, model systems (such as animal models) and methods are provided which utilize the unexpected different activation functions between and among the calcium channel subunit isoforms for the identification of novel agents, treatments, etc. useful in the modulation of conditions which arise from or manifest differences in action potential neurotransmitter release, voltage-dependent calcium channel activation, and so on. For example, methods for the identification of agents which alter activation potential dependent neurotransmitter release are provided. The methods include selecting an agent which binds a calcium channel isoform having or lacking a IVS3-S4 ET exon as described herein, and determining calcium channel activation or activation potential dependent neurotransmitter release in the presence and the absence of the agent. In some embodiments, candidate compounds may be screened by such methods. The methods also can include measurement of these parameters in other calcium channel subunits which manifest such differences in activation kinetics, including subunits in which an NP exon is added or is substituted for the ET exon.
Use of the foregoing compositions in the preparation of a medicament, and particularly in the preparation of a medicament for the treatment of stroke, pain (e.g., neuropathic pain), traumatic brain injury, or a condition which results from excessive or insufficient voltage regulated calcium influx, is provided.
These and other aspects of the invention are described in greater detail below.